CN108982684B - Method for detecting and identifying Gelidium amansii - Google Patents

Method for detecting and identifying Gelidium amansii Download PDF

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CN108982684B
CN108982684B CN201810668607.5A CN201810668607A CN108982684B CN 108982684 B CN108982684 B CN 108982684B CN 201810668607 A CN201810668607 A CN 201810668607A CN 108982684 B CN108982684 B CN 108982684B
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gelidium amansii
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刘圆
李伯超
李莹
黄艳菲
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Southwest Minzu University
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Abstract

The invention relates to a method for detecting ingredients of Gelidium amansii, which comprises the following steps: a method for detecting ingredients of Gelidium amansii, which is characterized by comprising the following steps: 1) preparing a test solution of Gege vegetable; 2) detecting the Gelidium amansii test solution by adopting an ultrahigh liquid chromatography-tandem mass spectrometry, namely an ultrahigh liquid chromatography-mass spectrometry spectrum of the test solution; 3) and comparing the ultrahigh liquid chromatography-mass spectrum of the test sample with a liquid chromatography-mass spectrum database, and qualitatively analyzing the components in the Gelidium amansii. The invention firstly establishes a UPLC-QTOF-MS/MS qualitative identification method for chemical components in the whole herb of cardamine violifolia (gelidium amansii) in cardamine plants, and identifies the chemical components in the whole herb of gelidium amansii through a total ion flow diagram, an element composition analysis diagram and a mass spectrogram and by combining a large number of references. Scientific experimental data and theoretical basis are provided for the deep research of the Gege vegetable.

Description

Method for detecting and identifying Gelidium amansii
Technical Field
The invention relates to a method for detecting and identifying Gelidium amansii.
Background
Cardamine L, belonging to Brassicaceae, has about 160 plants, mainly distributed in temperate regions, originated in plain, mountain, sand or fine stone land, mostly originated in the shade and damp place, and a little originated in calcareous soil. Resource survey shows that: the existing cardamine plants in 39 species in China account for about 30 percent of the total number of cardamine plants and are distributed in various places in the south and the north of China. Representative plants of Cardamine include Cardamine violaceum, Cardamine diffusa, Cardamine macrophylla, Cardamine sinica, Cardamine margarita, Cardamine hirsuta, Cardamine hupingshanensis, Cardamine cordifolia, etc. In the prior art, the separation research of chemical components of cardamine sinicus, cardamine sinicus and cardamine violacea produced in Henan is mainly focused.
Disclosure of Invention
The identification of main chemical components in plant medicines is a necessary means for deep research on unknown medicinal materials, and the detection and analysis of the components of the schizonepeta through the ultra-high liquid chromatography-tandem mass spectrometry is not found at present, and the research on the chemical components of the schizonepeta tenuifolia is not reported.
The invention provides a method for detecting and identifying Gelidium amansii by adopting an ultra-high liquid chromatography-tandem mass spectrometry method for the first time.
The scheme of the invention is as follows:
a method for detecting ingredients of Gelidium amansii, which is characterized by comprising the following steps:
1) preparing a test solution of Gege vegetable;
2) detecting the Gelidium amansii test solution by adopting an ultrahigh liquid chromatography-tandem mass spectrometry, namely an ultrahigh liquid chromatography-mass spectrometry spectrum of the test solution;
3) comparing the ultrahigh liquid chromatography-mass spectrum of the test sample with a liquid chromatography-mass spectrum database, and qualitatively analyzing components in Gege;
the conditions of the ultra-high liquid chromatography are as follows: using a C18 liquid chromatographic column; and performing gradient elution by using a mobile phase A formic acid aqueous solution and a mobile phase B acetonitrile, wherein the gradient elution procedure is as follows:
Figure BDA0001708477490000011
Figure BDA0001708477490000021
in an embodiment of the invention, the preparation method of the Gelidium amansii test solution comprises the following steps: mixing Gelidium amansii sample powder with methanol water solution, and extracting to obtain the Gelidium amansii sample solution. The following method can be used for preparation, but is not limited to the method: 0.5g of Gelidium amansii sample powder was precisely weighed, placed in a round-bottomed flask, and 50mL of 75% methanol solution was added to weigh the total weight. Heating and refluxing for 1.5h, cooling to room temperature, weighing the total weight, and supplementing the weight with 75% methanol solution to make it equal to the weight before refluxing. Shaking uniformly, filtering, and concentrating the filtrate. The concentrated filtrate was transferred to a 25mL volumetric flask and the volume was increased to the mark with 75% methanol solution. And (3) extracting a certain amount of the prepared solution by using an injector, filtering the solution by using a microporous filter membrane of 0.22 mu m for three times, and injecting the solution into a sample injection bottle to be used as a Gelidium sample solution for later use.
Wherein the extraction method comprises solvent extraction, ultrasonic extraction, microwave extraction, supercritical fluid extraction, microwave-assisted extraction, etc. The preparation method also comprises some conventional operations in the field, such as stirring, concentration, filtration, constant volume and the like.
In one embodiment of the invention, the specification of the chromatographic column is 2.1 × 100mm, 1.8 μm.
In an embodiment of the present invention, the content of formic acid in the aqueous formic acid solution is 0.05 to 0.3%, and preferably 0.1%.
In an embodiment of the present invention, the chromatographic conditions of the ultra-high performance liquid chromatography further include one or more of the following:
1) flow rate: 0.45-0.55mL.min-1(ii) a Preferably 0.5mL.min-1；
2) Column temperature: 33-36 ℃; preferably 35 ℃;
3) sample introduction amount: 1-2 μ L; preferably 1. mu.L.
In an embodiment of the invention, the ultra-high performance liquid chromatography-tandem mass spectrometry is ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry.
In an embodiment of the present invention, the mass spectrum conditions of the tandem mass spectrum include one or more of the following:
1) scanning range: 100-1500 Da;
2) temperature of the desolventizing gas: 3400 ℃.
In an embodiment of the present invention, the mass spectrum conditions of the tandem mass spectrum further include one or more of the following:
3) the ion scanning mode is: ESI-negative ion mode;
4) ion source temperature: 95-105 ℃; preferably 100 ℃;
5) flow rate of desolventizing gas: 800-850.L.h-1(ii) a Preferably 800L.h-1；
6) On-line calibration material: leucine Enkephalin (LE);
7) capillary voltage: 2.2-2.6 kV; preferably 2.5 kV;
8) taper hole voltage: 35-40V; preferably 40V;
9) low energy collision energy: 5.5-6.5V; preferably 6V;
10) high energy collision energy: 35-50V.
In one embodiment of the present invention, the component obtained by qualitative analysis at least comprises one of the following compounds: quercetin-rutinoside-hexoside, petunidin-3-coumaroyl-rutinoside-5-glucoside, sophoricoside, myricetin-3, 7-O-dirhamnoside, luteolin-7-O-rutinoside, luteolin-7-O-glucoside, 9-hydrogen peroxide-11- (3-amyl-2-epoxy ethyl) -10-undecylenic acid, 9, 10-epoxy-13-hydroxy-octadecenoic acid, 10-hydroxy-6, 8, 12-octadecenoic acid, 9, 10-epoxy-12-octadecenoic acid and linolenic acid.
On the basis of confirming the ingredients of the Gelidium amansii according to the qualitative analysis, the method can also carry out quantitative analysis on the Gelidium amansii ingredients, and comprises the following steps:
1) qualitatively analyzing to determine target components, and preparing reference substance solution from the target components; diluting the reference solution with multiple concentrations to obtain linear standard solutions with different concentrations, respectively injecting samples to record chromatograms, and drawing a standard curve to obtain a linear equation.
2) Detecting and recording the chromatogram of the reference solution, and comparing the chromatogram with the chromatogram of the test solution of Gelidium amansii to obtain the content of the target component.
Wherein the target component refers to the component of the content to be detected which is confirmed by qualitative analysis.
The calculation of the content of the target component may be performed by a conventional method such as an external standard method, an internal standard method, an area normalization method, or the like. When the internal standard method is used, the internal standard substance is selected to be a substance that does not interfere with the absorption peak of the target component, and can be obtained by the prior art and some routine experiments.
The invention also includes validity evaluations, including system applicability, specificity, quantitation limit, reproducibility, stability, and sample recovery, all of which are conventional methods, and can be varied by those skilled in the art within reasonable limits with reference to the prior art.
The invention also provides a method for identifying the Gelidium amansii, which comprises the following steps:
1) sampling to prepare a sample test solution;
2) detecting the sample test solution by adopting an ultrahigh liquid chromatography-tandem mass spectrometry, namely an ultrahigh liquid chromatography-mass spectrogram of the sample; the sample ultrahigh phase chromatogram-mass spectrogram comprises 11 characteristic peaks, wherein relative retention time of each characteristic peak is 0.47, 0.49, 0.54, 0.55, 0.57, 0.73, 0.82, 0.87, 0.90, 0.91 and 1 according to peak emergence time sequence by taking a peak corresponding to linolenic acid as a reference peak; the conditions of the ultra-high liquid chromatography are as follows: using a C18 liquid chromatographic column; and performing gradient elution by using a mobile phase A formic acid aqueous solution and a mobile phase B acetonitrile, wherein the gradient elution procedure is as follows:
Figure BDA0001708477490000031
Figure BDA0001708477490000041
the invention selects the linolenic acid with the peak number 14 as a reference peak, and can also select the peaks of other known compounds such as 8, 9 and the like as the reference peak, and correspondingly calculates the relative retention time, and no specific value is given here.
The following method can be used for preparation, but is not limited to the method: 0.5g of powder of a sample to be tested was precisely weighed, placed in a round-bottomed flask, and 50mL of 75% methanol solution was added to weigh the total weight. Heating and refluxing for 1.5h, cooling to room temperature, weighing the total weight, and supplementing the weight with 75% methanol solution to make it equal to the weight before refluxing. Shaking uniformly, filtering, and concentrating the filtrate. The concentrated filtrate was transferred to a 25mL volumetric flask and the volume was increased to the mark with 75% methanol solution. A certain amount of the prepared solution is extracted by a syringe, and is injected into a sample bottle as a sample solution for standby after passing through a 0.22 mu m microporous filter membrane for three times.
In one embodiment of the invention, the characteristic peaks with relative retention times of 0.44, 0.52, 0.56 and 1.03 are also included.
The invention has the beneficial effects that:
the invention relates to a method for qualitatively identifying chemical components in cardamine violacea (gelidium amansii) whole grass by establishing UPLC-QTOF-MS/MS in cardamine plants for the first time, and the chemical components in the gelidium amansii whole grass are identified by a total ion flow diagram, an element composition analysis diagram and a mass spectrogram and by combining a large number of references. Scientific experimental data and theoretical basis are provided for the deep research of the Gege vegetable.
Secondly, the experiment identifies the existence of linolenic acid in the whole herb of Gelidium amansii for the first time, and provides a new idea for the comprehensive development and utilization of Gelidium amansii.
Drawings
FIG. 1 is a total ion flow graph of a whole grass sample solution of Gelidium amansii;
figure 2 mass fragmentation diagram of compound 1;
figure 3 mass fragmentation diagram of compound 2;
figure 4 mass fragmentation diagram of compound 3;
figure 5 mass fragmentation diagram of compound 4;
figure 6 mass fragmentation diagram of compound 5;
figure 7 mass fragmentation diagram of compound 6;
figure 8 mass fragmentation diagram of compound 7;
figure 9 mass fragmentation diagram of compound 8;
figure 10 mass fragmentation diagram of compound 9;
figure 11 mass fragmentation diagram of compound 10;
figure 12 mass fragmentation diagram of compound 11;
figure 13 mass fragmentation diagram of compound 12;
figure 14 mass fragmentation diagram of compound 13;
figure 15 mass fragmentation diagram of compound 14;
figure 16 mass fragmentation diagram of compound 15.
Detailed Description
Instrument and material
1 apparatus
UPLC-QTOF-MS/MS (Waters ultra high performance liquid model: UPLC I Class, mass spectrometer model: XEVO G2-S); ultra-high performance liquid chromatograph: ACQUITY UPLC (Waters, USA, including quaternary high pressure gradient pump, vacuum degasser, autosampler, column oven, diode array detector, Empower chromatography workstation).
2 materials and reagents
The Shige vegetable sample is obtained from Qiang autonomous state and Ganzui autonomous state of Abaca of Sichuan province in 2016-2017, respectively, and is crushed and sieved with No. 3 sieve for later use.
Acetonitrile is chromatographically pure; the water is double distilled water; the rest reagents are analytically pure.
Second, test method
1 chromatographic conditions
A chromatographic column: ACQUITY UPLC HSS T3C 18 (2.1X 100mm, 1.8 μm); flow rate: 0.5mL.min-1(ii) a Column temperature: the sample injection amount is 1.0 mu L at 35 ℃, and an automatic sample injection mode is adopted.
Mobile phase: a (0.1% formic acid-water), B (acetonitrile), gradient elution with varying proportions of mobile phase as shown in Table 5-1
TABLE 5-1 chromatographic conditions
Table 5-1 Chromatographic conditions
Figure BDA0001708477490000051
Figure BDA0001708477490000061
2 parameters of Mass Spectrometry
The ion scanning mode is: ESI-negative ion mode; scanning range: 100-1500 Da; ion source temperature: 100 ℃; temperature of the desolventizing gas: 400 ℃; flow rate of desolventizing gas: 800.L.h-1(ii) a On-line calibration material: leucine Enkephalin (LE); capillary voltage: 2.5 kV; taper hole voltage: 40V; low energy collision energy: 6V; high energy collision energy: 35-50V.
Data acquisition software; MassLynx4.1; the data acquisition mode is as follows: MSE.
3 preparation of test solution
0.5g of Gelidium amansii sample powder was precisely weighed, placed in a round-bottomed flask, and 50mL of 75% methanol solution was added to weigh the total weight. Heating and refluxing for 1.5h, cooling to room temperature, weighing the total weight, and supplementing the weight with 75% methanol solution to make it equal to the weight before refluxing. Shaking uniformly, filtering, and concentrating the filtrate. The concentrated filtrate was transferred to a 25mL volumetric flask and the volume was increased to the mark with 75% methanol solution. And (3) extracting a certain amount of the prepared solution by using an injector, filtering the solution by using a microporous filter membrane of 0.22 mu m for three times, and injecting the solution into a sample injection bottle to be used as a Gelidium sample solution for later use.
Third, results and analysis
The whole herb sample solution of Gege is scanned in ion mode. And (3) according to the outflow sequence of peaks in the total ion flow diagram, an ion loading mode and MSE data, detecting the compound in the Gelidium amansii whole grass by adopting MassLynx4.1 software in a combined manner. By analyzing the characteristic fragment ion characteristics in the mass spectrogram and comparing the chromatographic retention time (T/min) and the relative molecular mass [ M + H ] of a comparative compound]+Chemical Formula (Formula) and fragment ions (m/z), and numerous references to extrapolate. And carrying out primary analysis on the compounds in the whole herb of Gelidium amansii. The total ion flow diagram is shown in figure 1. The results of the identification and analysis of the chemical components in the negative ion mode are shown in Table 1.
TABLE 1 identification and analysis of chemical composition of Gege vegetables
Figure BDA0001708477490000062
Figure BDA0001708477490000071
The compound formula is shown in the sLynx4.1 database as follows: c33H43O21. Obtaining chromatographic peak with retention time of 4.06min in negative ion mode, wherein the M/z of parent nucleus ion of the compound detected in MS/MS spectrum is 771.1996[ M + H ]]-And characteristic fragment ions m/z of 179.0364, 327.0750, fragment ions m/z of 179 correspond to hexoses. The study of Lu ocean, etc. shows that the compound with the number 2 and the quercetinThe m/z 771 of rutinoside-hexaglycoside, the characteristic fragment ion m/z 179 and the molecular formula are the same, it can be concluded that the compound is quercetin-rutinoside-hexaglycoside. The mass spectrum is shown in figure 3.
Compound No. 3 compound showed compound formula in MassLynx4.1 database: c35H49O29. Obtaining chromatographic peak with retention time of 4.23min in negative ion mode, wherein the M/z of parent nucleus ion of the compound detected in MS/MS spectrum is 933.2329[ M + H ]]-And the characteristic fragment ions m/z 771.1971, 934.2358, 1095.2859, the fragment ion m/z 771 corresponds to de-1-molecule anhydroglucose. The Zhaojing et al study showed that compound No. 3 was 933 for m/z, characteristic fragment ions m/z 771, 934, 1095 and the same molecular formula as petunidin-3-coumaroyl-rutinosyl-5-glucoside, and the compound was concluded to be petunidin-3-coumaroyl-rutinosyl-5-glucoside. The mass spectrum is shown in figure 4.
Compound No. 5 compound showed compound formula in MassLynx4.1 database: c27H29O16. Obtaining chromatographic peak with retention time of 4.63min in negative ion mode, wherein the M/z of parent nucleus ion of the compound detected in MS/MS spectrum is 609.1456[ M + H ]]-And characteristic fragment ions m/z 271.0243, 299.0178. Zhao Fengchun, etc[33]Research shows that the compound No. 5 and the sophoricoside have m/z609, characteristic fragment ions m/z 271, 299 and the same molecular formula, and the compound can be concluded to be the sophoricoside. The mass spectrum is shown in figure 6.
Compound No. 6 compound showed compound formula in MassLynx4.1 database: c22H29O16. Obtaining chromatographic peak with retention time of 4.72min in negative ion mode, wherein the M/z of parent nucleus ion of the compound detected in MS/MS spectrum is 609.1450[ M + H ]]-And characteristic fragment ions m/z 174.9555, 467.1574, wherein the fragment ion m/z 467 corresponds to a fragment ion formed by removing rhamnose from rutin. Studies such as Nippon glorybower et al show that the compound with the number 6 is the same as myricetin-3, 7-O-dirhamnoside M/Z609, characteristic fragment ions M/Z175 and 467 and molecular formula, and the compound is inferred to be myricetin-3, 7-O-dirhamnoside. The mass spectrum is shown in figure 7.
Compound No. 8 compound showed compound formula in MassLynx4.1 database: c27H29O15. Obtaining chromatographic peak with retention time of 4.89min under negative ion mode, wherein the M/z of parent nucleus ion of the compound detected in MS/MS spectrum is 593.1509[ M + H ]]-And characteristic fragment ion m/z 447.0915, fragment ion m/z447 corresponds to the parent nucleus ion obtained by losing one molecule of glucose and one molecule of rhase. Yangying et al[35]Research shows that the compound No. 8 and luteolin-7-O-rutinoside have the m/z of 593, the characteristic fragment ion m/z447 and the same molecular formula, and the compound is concluded to be luteolin-7-O-rutinoside. The mass spectrum is shown in figure 9.
Compound No. 9 compound showed compound formula in MassLynx4.1 database: c21H19O11. Obtaining chromatographic peak with retention time of 6.26min in negative ion mode, wherein the M/z of parent nucleus ion of the compound detected in MS/MS spectrum is 447.0930[ M + H ]]-And characteristic fragment ion m/z 174.9561, fragment ion m/z 175 corresponding to the parent nucleus ion by losing one molecule of glucose. Studies of Zqian and the like show that the compound with the code 9 is the same as the luteolin-7-O-glucoside M/Z447, the characteristic fragment ion M/Z175 and the molecular formula, and the compound can be concluded to be the luteolin-7-O-glucoside. The mass spectrum is shown in figure 10.
Compounds No. 10, 11, 12, 13, 14 showed the compound formula in the MassLynx4.1 database, respectively: c18H31O5、C18H31O4、C18H29O3、C18H31O3、C18H29O2. Obtaining chromatographic peaks with retention time of 7.03min, 7.46min, 7.71min, 7.85min and 8.55min respectively in negative ion mode, wherein the M/z of parent nucleus ion of the compound detected in MS/MS spectrum is 327.2171[ M + H ] respectively]-、311.2224[M+H]-、293.2117[M+H]-、295.2277[M+H]-、277.2174[M+H]-. The research of aging sensitivity and the like shows that mass spectrum data can be possibly combined after being retrieved by ChemSepder web DatabaseAnd (4) performing conjecture. These five compounds are known to be fatty acids, in the order: 9-hydroperoxide-11- (3-amyl-2-epoxy ethane) -10-undecylenic acid, 9, 10-epoxy-13-hydroxy-octadecenoic acid, 10-hydroxy-6, 8, 12-octadecenoic acid, 9, 10-epoxy-12-octadecenoic acid and linolenic acid. The mass spectrograms are shown in figures 11, 12, 13, 14 and 15.
The compound is obtained by separating cardamine hirsute and cardamine yedoensis for the first time.
The mass spectra of unidentified compounds 1, 4, 7 and 15 are shown in the attached figures 2, 5, 8 and 16, respectively.
The test establishes an UPLC-QTOF-MS/MS analysis method for qualitatively identifying compounds in the whole herb of cardamine violifolia (gelidium amansii) in cardamine plants for the first time, and comprehensively and systematically researches the gelidium amansii. By referring to a large amount of literature data, 11 compounds in the whole herb of Gelidium amansii are identified by comparing compound retention time, accurate molecular weight and fragment ion information, wherein 6 flavonoid compounds are respectively: quercetin-rutinoside-hexoside, petunidin-3-coumaroyl-rutinoside-5-glucoside, sophoricoside, myricetin-3, 7-O-dirhamnoside, luteolin-7-O-rutinoside, and luteolin-7-O-glucoside; 5, the fatty acid compounds are respectively: 9-hydroperoxide-11- (3-amyl-2-epoxy ethane) -10-undecylenic acid, 9, 10-epoxy-13-hydroxy-octadecenoic acid, 10-hydroxy-6, 8, 12-octadecenoic acid, 9, 10-epoxy-12-octadecenoic acid and linolenic acid. Provides a basis for the systematic research of the chemical components of the Gelidium amansii in the later period.
The U.S. FDA study demonstrated that: the lack of linolenic acid can lead to the growth retardation of the brain and retina of children, the inability to concentrate attention, unbalanced nutrition and the inability to effectively absorb nutrient components, and directly lead to more than 30 symptoms and diseases such as intelligence growth retardation, uncoordinated movement, weak eyesight, hyperactivity, obesity, anorexia, slow growth, low immunity and the like. The experiment identifies the existence of linolenic acid in the whole herb of Gelidium amansii for the first time, and provides a new idea for the comprehensive development and utilization of Gelidium amansii.
Although the present invention has been described herein with reference to the illustrated embodiments thereof, the above-described embodiments are only one of the preferred embodiments of the present invention, and the embodiments of the present invention are not limited thereto, and it should be understood that many other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure.

Claims (13)

1. A method for detecting ingredients of Gelidium amansii, which is characterized by comprising the following steps:
1) preparing a test solution of Gege vegetable;
2) detecting the Gelidium amansii test solution by using an ultra-high liquid chromatography-tandem mass spectrometry to obtain an ultra-high liquid chromatography-mass spectrogram of the test solution;
3) comparing the ultrahigh liquid chromatography-mass spectrum of the test sample with an ultrahigh liquid chromatography-mass spectrum database, and qualitatively analyzing components in Gege;
the conditions of the ultra-high liquid chromatography are as follows: using a C18 liquid chromatographic column; and performing gradient elution by using a mobile phase A formic acid aqueous solution and a mobile phase B acetonitrile, wherein the gradient elution procedure is as follows:
Figure FDA0002952282540000011
；
the components obtained by qualitative analysis at least comprise one of the following compounds: quercetin-rutinoside-hexoside, petunidin-3-coumaroyl-rutinoside-5-glucoside, sophoricoside, myricetin-3, 7-O-dirhamnoside, luteolin-7-O-rutinoside, luteolin-7-O-glucoside, 9-hydrogen peroxide-11- (3-amyl-2-epoxy ethyl) -10-undecylenic acid, 9, 10-epoxy-13-hydroxy-octadecenoic acid, 10 hydroxy-6, 8, 12-octadecenoic acid, 9, 10-epoxy-12-octadecenoic acid, and linolenic acid.
2. The method of detecting gelidium amansii ingredients as claimed in claim 1, wherein: the preparation method of the Gelidium amansii test solution comprises the following steps: mixing Gelidium amansii sample powder with methanol water solution, and extracting to obtain the Gelidium amansii sample solution.
3. The method of detecting gelidium amansii ingredients as claimed in claim 1, wherein: the specification of the chromatographic column is 2.1X 100mm and 1.8 mu m.
4. The method of detecting gelidium amansii ingredients as claimed in claim 1, wherein: the content of formic acid in the aqueous solution of formic acid is 0.05-0.3%.
5. The method of detecting Gelidium amansii ingredients of claim 4, wherein: the content of formic acid in the aqueous formic acid solution is 0.1 percent.
6. The method of detecting gelidium amansii ingredients as claimed in claim 1, wherein: the chromatographic conditions of the ultra-high liquid chromatography further comprise one or more of:
1) flow rate: 0.45-0.55 mL/min-1；
2) Column temperature: 33-36 ℃;
3) sample introduction amount: 1-2. mu.L.
7. The method of detecting Gelidium amansii ingredients of claim 6, wherein: the chromatographic conditions of the ultra-high liquid chromatography further comprise one or more of:
1) flow rate: 0.5 mL/min-1；
2) Column temperature: 35 ℃;
3) sample introduction amount: 1 μ L.
8. The method of detecting gelidium amansii ingredients as claimed in claim 1, wherein: mass spectral conditions of the tandem mass spectrum include one or more of:
1) scanning range: 100-1500 Da;
2) temperature of the desolventizing gas: at 400 ℃.
9. The method of detecting gelidium amansii ingredients as claimed in claim 8, wherein: the mass spectrometry conditions of the tandem mass spectrometry further comprise one or more of:
3) the ion scanning mode is: ESI-negative ion mode;
4) ion source temperature: 95-105 ℃;
5) flow rate of desolventizing gas: 800-850 L.h-1；
6) On-line calibration material: leucine enkephalin LE;
7) capillary voltage: 2.2-2.6 kV;
8) taper hole voltage: 35-40V;
9) low energy collision energy: 5.5-6.5V;
10) high energy collision energy: 35-50V.
10. The method of detecting gelidium amansii ingredients of claim 9, wherein: the mass spectrometry conditions of the tandem mass spectrometry further comprise one or more of:
3) the ion scanning mode is: ESI-negative ion mode;
4) ion source temperature: 100 ℃;
5) flow rate of desolventizing gas: 800L.h-1；
6) On-line calibration material: leucine enkephalin LE;
7) capillary voltage: 2.5 kV;
8) taper hole voltage: 40V;
9) low energy collision energy: 6V;
10) high energy collision energy: 35-50V.
11. A method of identifying geigera, comprising:
1) sampling to prepare a sample test solution;
2) detecting the sample test solution by adopting an ultrahigh liquid chromatography-tandem mass spectrometry, namely an ultrahigh liquid chromatography-mass spectrogram of the sample;
the sample ultrahigh liquid chromatography-mass spectrogram comprises 11 characteristic peaks, wherein relative retention time of each characteristic peak is 0.47, 0.49, 0.54, 0.55, 0.57, 0.73, 0.82, 0.87, 0.90, 0.91 and 1 according to peak emergence time sequence by taking a peak corresponding to linolenic acid as a reference peak; the conditions of the ultra-high liquid chromatography are as follows: using a C18 liquid chromatographic column; and performing gradient elution by using a mobile phase A formic acid aqueous solution and a mobile phase B acetonitrile, wherein the gradient elution procedure is as follows:
Figure FDA0002952282540000031
12. the method of identifying Gelidium amansii as claimed in claim 11, wherein: the compounds with relative retention time of 0.47, 0.49, 0.54, 0.55, 0.57, 0.73, 0.82, 0.87, 0.90, 0.91 and 1 corresponding to characteristic peaks are quercetin-rutinoside-hexoside, petunidin-3-coumaroyl rutinoside-5-glucoside, sophorafenone glycoside, myricetin-3, 7-O-dirhamnoside, luteolin-7-O-rutinoside, luteolin-7-O-glucoside, 9-hydrogen peroxide-11- (3-amyl-2-epoxyethyl) -10-undecylenic acid, 9, 10-epoxy-13-hydroxy-octadecenoic acid, 10-hydroxy-6, 8, 12-octadecenoic acid, 9, 10-epoxy-12-octadecenoic acid, and 1-methyl-beta-hydroxy-5-O-glucoside, respectively, Linolenic acid.
13. The method of identifying Gelidium amansii as claimed in claim 11, wherein: also included are characteristic peaks at relative retention times of 0.44, 0.52, 0.56, 1.03.
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